From the Department of Medicine, Huddinge Karolinska Institutet, Stockholm, Sweden DYNAMICS OF NATURAL KILLER CELL HOMEOSTASIS – IMPLICATIONS FOR CELL-BASED CANCER IMMUNOTHERAPY Aline Pfefferle Stockholm 2019 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by E-Print AB 2019 © Aline Pfefferle, 2019 ISBN 978-91-7831-434-8 DYNAMICS OF NATURAL KILLER CELL HOMEOSTASIS – IMPLICATIONS FOR CELL-BASED CANCER IMMUNOTHERAPY THESIS FOR DOCTORAL DEGREE (Ph.D.) By Aline Pfefferle Public defence: Wednesday 5th of June, 2019 at 9:30 am Lecture Hall 4V, Alfred Nobels Allé 8, Karolinska Institutet, Huddinge Principal Supervisor: Opponent: Professor Karl-Johan Malmberg Thierry Walzer, PhD Karolinska Institutet INSERM Department of Medicine, Huddinge Centre international de recherche en infectiologie Center for Infectious Medicine Examination Board: Co-supervisor(s): Docent Lisa Westerberg Ebba Sohlberg, PhD Karolinska Institutet Karolinska Institutet Department of Microbiology, Tumor and Cell Department of Medicine, Huddinge Biology Center for Infectious Medicine Docent Petter Brodin Karolinska Institutet Department of Women’s and Children’s Health Science for Life Lab Docent Fredrik Bergh Thorén Göteborgs Universitet Sahlgrenska Cancer Center To my family ABSTRACT Natural killer (NK) cells comprise a central role within the innate immune system, eliminating virally infected, foreign and transformed cells through their natural cytotoxic capacity. Release of their cytotoxic granules is tightly controlled through the balance of a large repertoire of inhibitory and activating receptors, and it is the unique combination of these receptors on individual cells that confers them their immense diversity both in phenotype and functionality. This thesis aimed to investigate the mechanisms sustaining NK cell homeostasis with the aim of translating these findings into more efficient NK cell-based immunotherapies against cancer. In paper I, we set out to define a transcriptional timeline for NK cell differentiation through the use of single-cell RNA sequencing of unique differentiation subsets ranging from CD56bright to adaptive NKG2C+CD56dim NK cells. Transcriptional differentiation was concentrated within the surprisingly diverse CD56bright subset which gradually transitioned into CD56dim NK cells before terminal differentiation into adaptive CD56dim NK cells. The vastly diverse yet unique NK cell repertoire within an individual is surprisingly stable over time considering the constant renewal of these cells at steady state. In paper II, we performed an in-depth analysis of homeostatic proliferation in human NK cells. We identified a high degree of intra-lineage plasticity combined with transcriptional reprogramming associated with the acquired phenotype as the underlying mechanisms maintaining repertoire stability at steady state. In paper III, we examined the role of NK cells in a setting of perturbed homeostasis, namely patients with high-risk myelodysplastic syndrome undergoing immunomodulatory treatment with 5-azacytidine. We identified a role for 5-azacytidine in modifying the global NK cell repertoire, as uptake of the drug by proliferating NK cells resulted in increased expression of killer cell immunoglobulin-like receptors (KIR) and improved functionality. In paper IV we identified a dose-dependent cytokine addiction in IL-15 expanded NK cells, leading to the induction of apoptosis upon cytokine withdrawal. A proliferation-dependent induction of the short splice variant of BIM, combined with an altered BCL-2/BIM ratio resulted in sensitization to cell death post withdrawal. This thesis provides new insights into the dynamic nature of NK cell homeostasis, from understanding NK cell differentiation at the transcriptional level to perturbations after cytokine stimulation and immunomodulatory therapies. LIST OF SCIENTIFIC PAPERS I. Pfefferle A*, Netskar H*, Ask EH, Lorenz S, Sohlberg E, Clancy T‡, Malmberg KJ‡. A temporal transcriptional map of human natural killer cell differentiation. Manuscript. II. Pfefferle A, Jacobs B, Ask EH, Lorenz S, Clancy T, Goodridge JP, Sohlberg E, Malmberg KJ. Intra-lineage plasticity and functional reprogramming maintain natural killer cell repertoire diversity. BioRxiv. 2019. Manuscript. III. Sohlberg E, Pfefferle A, Andersson S, Baumann BC, Hellström-Lindberg E, Malmberg KJ. Imprint of 5-azacytidine on the natural killer cell repertoire during systemic treatment for high-risk myelodysplastic syndrome. Oncotarget. 2015 Oct 27;6(33):34178-34190. IV. Jacobs B, Pfefferle A, Clement D, Berg-Larsen A, Sætersmoen ML, Lorenz S, Wiiger MT, Goodridge JP, Malmberg KJ. Induction of the BIM short splice variant sensitizes proliferating NK cells to IL-15 withdrawal. Journal of Immunology. 2019 Feb 1; 202(3):736-746. *‡ These authors have contributed equally. Paper IV is Copyright Ó 2019 The American Association of Immunologists, Inc. LIST OF ADDITIONAL RELEVANT PUBLICATIONS I. Goodridge JP, Jacobs B, Sætersmoen ML, Clement D, Hammer Q, Clancy T, Skarpen E, Brech A, Landskron J, Grimm C, Pfefferle A, Meza-Zepeda L, Lorenz S, Wiiger MT, Louch WE, Ask EH, Liu LL, Oie VYS, Kjällquist U, Linnarsson S, Patel S, Taskén K, Stenmark H, Malmberg KJ. Remodeling of secretory lysosomes during education tunes functional potential in NK cells. Nature Communications. 2019 Jan 31;10(1):514. II. Liu LL, Béziat V, Oie VYS, Pfefferle A, Schaffer M, Lehmann S, Hellström- Lindberg, E, Söderhäll S, Heyman M, Grander D, Malmberg KJ. Ex vivo expanded adaptive NK cells effectively kill primary acute lymphoblastic leukemia cells. Cancer Immunology Research. 2017 Aug;5(8):654-665. III. Liu LL, Pfefferle A, Oie VYS, Björklund AT, Béziat V, Goodridge JP, Malmberg KJ. Harnessing adaptive natural killer cells in cancer immunotherapy. Molecular Oncology. 2015 Dec;9(10):1904-1917. CONTENTS 1 Introduction .................................................................................................................... 1 1.1 Basic Concepts of NK Cell Biology .................................................................... 1 1.1.1 NK cell development ............................................................................... 2 1.1.2 NK cell killing .......................................................................................... 2 1.1.3 NK cell receptors ..................................................................................... 4 1.1.4 NK cell differentiation ............................................................................. 7 1.1.5 NK cell homeostasis ................................................................................ 8 1.1.6 NK cell education .................................................................................. 10 1.2 NK Cells in the Disease Setting ......................................................................... 11 1.2.1 Myelodysplastic syndrome .................................................................... 11 1.2.2 Adoptive NK cell therapy ...................................................................... 13 2 Aims ............................................................................................................................. 16 3 Results and Discussion ................................................................................................ 17 3.1 NK Cell Differentiation ..................................................................................... 17 3.1.1 The regulome of human NK cell differentiation as we knew it ............ 17 3.1.2 A temporal transcriptional map of NK cell differentiation ................... 17 3.1.3 The bridge connecting CD56bright to CD56dim NK cells ........................ 19 3.1.4 Formation of the functional template for education .............................. 20 3.1.5 Methodological considerations for scRNA-seq analysis ...................... 20 3.2 NK Cell Homeostasis ......................................................................................... 21 3.2.1 Subset repertoire stability ...................................................................... 22 3.2.2 Perturbations in the disease setting ........................................................ 25 3.2.3 Implications for immunotherapy ........................................................... 27 4 Concluding Remarks .................................................................................................... 32 5 Future Outlook ............................................................................................................. 33 6 Acknowledgements ...................................................................................................... 34 7 References .................................................................................................................... 37 LIST OF ABBREVIATIONS 5-aza 5-azacytidine ADCC Antibody-dependent cellular cytotoxicity AML Acute myeloid leukemia ATAC Assay for transposase-accessible chromatin BiKE Bi-specific killer engager BIM S BIM short (splice variant) BM Bone marrow CAR Chimeric antigen receptor CCL C-C chemokine ligand CCR C-C chemokine receptor CD Cluster of differentiation cDNA Complementary DNA ChIP Chromatin immunoprecipitation CIS Cytokine induced SH2-containing protein CLP Common lymphoid progenitor CMP Common myeloid progenitor CMV Cytomegalovirus CR Complete remission CXCR C-X-C chemokine receptor DC Dendritic cell DNA Deoxyribonucleic acid DNAM-1 DNAX accessory molecule-1 DR Death receptor